Methods And Devices For Amplification Of Nucleic Acid

ABSTRACT

The present invention relates to methods and devices for amplifying nucleic acid, and, in particular, amplifying so as to generate products on a surface without the use of emulsions. In a preferred embodiment, a plurality of groups of amplified product are generated on the surface, each group positioned in different (typically predetermined) locations on said surface so as to create an array.

FIELD OF THE INVENTION

The present invention relates to methods and devices for amplifyingnucleic acid, and, in particular, amplifying so as to generate productson a surface without the use of emulsions. In a preferred embodiment, aplurality of groups of amplified product are generated on the surface,each group positioned in different locations on said surface so as tocreate an array.

BACKGROUND

The use of water-in-oil emulsions was recently adapted to biologicalapplications by several groups aiming at improving enzymes by directedevolution, SNP analysis on streptavidin coated beads, and DNAsequencing. Dressman et al., “Transforming single DNA molecules intofluorescent magnetic particles for detection and enumeration of geneticvariations” Proc Natl Acad Sci USA, 100:8817-8822 (2003); Ghadessy etal., “Directed evolution of polymerase function by compartmentalizedself replication” Proc Natl Acad Sci USA 98:4552-4557 (2001); Tawfik etal., “Man-made cell-like compartments for molecular evolution”16:652-656 (1998); Margulies et al., “Genome sequencing inmicrofabricated high-density picolitre reactors” Nature 437:376-80(2005); and Shendure et al., “Accurate multiplex polony sequencing of anevolved bacterial genome” Science 309:1728-1732 (2005).

Unfortunately, the emulsion techniques are slow and cumbersome. As aresult, they are not ideal for commercial applications.

SUMMARY OF THE INVENTION

The present invention relates to methods and devices for amplifyingnucleic acid, and, in particular, amplifying so as to generate productson a surface without the use of emulsions. In a preferred embodiment, aplurality of groups of amplified product are generated on the surface,each group positioned in different (typically predetermined) locationson said surface so as to create an array. In one embodiment, each groupis homogeneous. In one embodiment, each group consists of amplifiedproduct of a single nucleic acid template. In one embodiment, the methodcomprises performing limiting dilution PCR within closed compartments(e.g. sealed regions) created by two surfaces coming together.

In one embodiment, the present invention contemplates a method formaking an array, comprising: a) providing: a first element comprising afirst surface; a second element comprising a second surface; a pluralityof indentations, wherein said indentations are on either said firstsurface or said second surface (or on both surfaces); and a solutioncomprising molecules selected from the group consisting of biomoleculesand anchoring molecules; b) contacting said first surface with saidsolution under conditions such that at least a portion of said moleculesattach to at least a portion of said first surface so as to create amodified surface comprising attached molecules; and c) positioning saidsecond surface on top of said modified surface, so as to create aplurality of first regions defined by said indentations, said firstregions comprising unmasked attached molecules, and second regionscomprising masked attached molecules, thereby making an array. Thepresent invention contemplates this embodiment of an array as a device.

It is not intended that the present invention be limited by theplacement of the biomolecules and/or anchoring molecules. In oneembodiment, said contacting of step b) causes the anchoring molecules tocontact the indentations, the flat surface, or both. Similarly, thebiomolecules may contact the indentations, the flat surface, or both.

In one embodiment, said positioning of step c) causes at least a portionof said solution on said first surface to move off of said firstsurface. In other words, bringing the surfaces into contact can causeliquid to be put under pressure so that some portion of the solutionvolume moves off (e.g. drains off and that portion of the volume isremoved or lost). It is not intended that the present invention belimited to how the solution is brought into contact with the surface instep b). In one embodiment, the surface is dipped or immersed in thesolution.

In one embodiment, said indentations have attached PCR primer(s) andsaid solution comprises nucleic acid template. In one embodiment, saidtemplate has been diluted to a concentration such that less than onehundred molecules on average (more preferably, less than 10, still morepreferably, less than 1 molecule) of template are in contact with anyone indentation. In one embodiment (e.g. where it is desired to havefewer empty wells), said template has been diluted to a concentrationsuch that less than three on average—but more than 1 molecule—oftemplate are in contact with any one indentation (or are within any oneregion). In one embodiment, said solution also contains reagents for PCR(e.g. polymerase, dNTPs, buffer, etc.). In a preferred embodiment, thetemplate amplified in any one sealed region is homogeneous (i.e. onlyone type of template was amplified). In a preferred embodiment,different (diluted) template is introduced into at least two differentsealed regions such that each of said different template issimultaneously amplified and each of said amplified product ishomogeneous.

It is not intended that the present invention be limited by theparticular geometry of the surfaces. In one embodiment, said firstsurface is substantially flat (e.g. flat over 90% of the surface orcomprising less than 10% deviation from flat). It can, but need not be,completely flat. It can be curved or only slightly curved. In oneembodiment, said contacting of step b) results in substantially theentire first surface being contacted with said solution.

It is not intended that the present invention be limited by the natureof the surfaces. In one embodiment, said first surface comprises glass.In one embodiment, said first surface comprises a surface of amicroscope slide. In one embodiment, said first surface comprises asurface of a microchip (e.g. a silicon surface). In a preferredembodiment, one or both surfaces comprise a polymer. The use of anelastomer is believed to enhance the sealing of one surface against theother.

It is not intended that the present invention be limited by the natureof the indentations. The term “indentation” as used herein, refers to aspace, cavity, dent, crater, well, depression, hollow, recess orimpression that is formed in the surface. In a preferred embodiment,indentations do not extend through the entire thickness of a surface.Useful dimensions for indentations are between 0.001 and 10,000 micronsin diameter, with depths between 0.1× and 10× (i.e. 10 times) thediameter, and spacing (separation) of between 1× and 3× (i.e. threetimes) the diameter. Typically, indentations are between 50 nanometersand 50 microns in diameter, with depths approximately 0.5× the diameter,and spacing approximately 1.2× and 2× (i.e. two times) the diameter. Forexample, in one embodiment, the present invention contemplatesindentations of 50 nanometers in diameter, that are 25 nanometers deep,and that are spaced 60 nanometers apart. In another embodiment, thepresent invention contemplates indentations of 5 microns in diameter,which are 2.5 microns deep, and spaced 7 microns apart.

While a hole can be an indentation, the hole preferably does not extendcompletely through the surface. In a preferred embodiment, each of saidindentations has a depth that extends up to the midpoint of said firstor second element (i.e. the depth of the indentation is equal to or lessthan one-half the thickness of the surface). In one embodiment, saidsecond surface is crenellated and the gaps comprise said indentations.In another embodiment, the indentations have raised edges. The term“raised edge” means that the edge of the indentation rises above theplane of the surface. In one embodiment, there are particles in theindentations (e.g. beads).

It is not intended that the present invention be limited by the mannerin which the indentations are manufactured. In one embodiment, theindentations are introduced into the surface by treating the surface(e.g. etching a surface of glass, silicon or otherwise etchablesurface). In another embodiment, the indentations are introduced bycasting or molding. In a preferred embodiment, the indentations areintegrally molded using a polymeric surface (e.g. plastic). The term“integrally molding” as used herein refers to the method of casting suchthat features are of unitary construction. The term “unitaryconstruction” refers to an association of elements (e.g. the surface andthe indentations) such that they are formed from the same piece of rawmaterial without the need for further integration. In one embodiment,the first surface comprises plastic and has indentations. In oneembodiment, said first surface is elastomeric.

In one embodiment, said indentations are evenly spaced. In oneembodiment, said attached molecules after step b) are attached oversubstantially the entire first surface at a substantially even density.In another embodiment, the molecules are attached randomly over thesurface or a portion of the surface.

It is not intended that the present invention be limited by the natureof the molecules. In one embodiment, said molecules are biomolecules. Ina preferred embodiment, said biomolecules are nucleic acid molecules(e.g. oligonucleotides, polynucleotides, etc.). In a particularlypreferred embodiment, said nucleic acid molecules comprise PCR primers.In another embodiment, said nucleic acid molecules comprise probes forRCA. In yet another embodiment, said nucleic acid molecules comprisehairpins (e.g. the hairpin allows the nucleic acid to self-prime in anextension reaction with a polymerase).

The present invention contemplates a variety of orientations for the twosurfaces. The two surfaces can be placed on edge (e.g. two microscopeslides, or a microscope slide and a cover, can be brought together sothat each contacts the other on the broad flat surface, but then can beplaced on edge) for purposes of detection in a device. In oneembodiment, after step (c) said first surface faces up, and said secondsurface faces down.

In another embodiment, the present invention contemplates a method ofamplifying nucleic acid template, comprising: a) providing: i) a firstelement comprising a first surface; ii) a second element comprising asecond surface; iii) a plurality of indentations, wherein saidindentations are on said first surface, second surface or both; iv) afirst solution comprising a plurality of first PCR primers; v) a secondsolution comprising a plurality of second PCR primers; and vi) a thirdsolution comprising template, polymerase, dNTPs and primers, whereinsaid primers are first PCR primers, second PCR primers or both; b)contacting said first surface with said first solution under conditionssuch that at least a portion of said first PCR primers attach to atleast a portion of said surface so as to create a first modified surfacecomprising attached molecules; c) contacting said second surface withsaid second solution under conditions such that at least a portion ofsaid second PCR primers attach to at least a portion of said bottomsurface so as to create a second modified surface comprising attachedmolecules; and d) positioning said second modified surface on top ofsaid first modified surface, so as to create a plurality of firstregions defined by said indentations, said first regions comprisingunmasked attached molecules, and second regions comprising maskedattached molecules, thereby making an array. In one embodiment, saidpositioning of step d) causes at least a portion of said solution onsaid surface of said first element to move off of said surface. In oneembodiment, each of said indentations has a depth that extends up to themidpoint between said bottom and top surfaces. In one embodiment, saidsecond surface is crenellated and the gaps comprise said indentations.In one embodiment, said indentations are wells. In one embodiment, saidfirst surface is substantially flat (10% or less deviation from flat).In one embodiment, said second surface comprises indentations. In oneembodiments, said indentations comprise particles (e.g. beads). In oneembodiment, said contacting of step b) results in substantially theentire surface being contacted with said solution. In one embodimentsaid indentations of said second surface element are evenly spaced. Inone embodiment, said attached molecules after step b) are attached oversubstantially the entire surface at a substantially even density. In oneembodiment, said first element comprises glass. In one embodiment, saidfirst surface comprises plastic. In one embodiment, said first surfacecomprises indentations. In one embodiment, said indentations are formedat the time said plastic surface is molded. In one embodiment, saidfirst surface is elastomeric. In one embodiment, said first element is amicroscope slide. In one embodiment, said first element is a microchip.In one embodiment, said first PCR primers consist of forward PCRprimers. In one embodiment, said first PCR primers consist of reversePCR primers. In one embodiment, said second PCR primers consist ofreverse PCR primers. In one embodiment, said second PCR primers consistof forward PCR primers.

In one embodiment of the above-described method, said template of saidthird solution has been diluted to a concentration such that less thanone hundred molecules on average (more preferably, less than 10, stillmore preferably, less than 1 molecule) of template are in contact withany one indentation. In a preferred embodiment, the template amplifiedin any one sealed region is homogeneous (i.e. only one type of templatewas amplified). In a preferred embodiment, different (diluted) templateis introduced into at least two different sealed regions such that eachof said different template is simultaneously amplified and each of saidamplified product is homogeneous. In one embodiment (e.g. where it isdesired to have fewer empty wells), said template has been diluted to aconcentration such that less than three on average—but more than 1molecule—of template are in contact with any one indentation (or arewithin any one region). In such a case, the amplified product may notalways be homogeneous (e.g. it may be the product of two differenttemplates).

In yet another embodiment, the present invention contemplates a methodof amplifying nucleic acid template, comprising: a) providing: i) afirst element comprising a first surface; ii) a second elementcomprising a second surfaces; iii) a plurality of indentations, whereinsaid indentations are on said first surface, second surface or both; iv)a first solution comprising a plurality of first and second PCR primers;v) a second solution comprising template, polymerase, dNTPs and primers,wherein said primers are first PCR primers, second PCR primers or both;b) contacting said first surface with said first solution underconditions such that at least a portion of said PCR primers attach to atleast a portion of said first surface so as to create a modified surfacecomprising attached molecules; and c) positioning said second surface ontop of said modified surface, so as to create a plurality of firstregions defined by said indentations, said first regions comprisingunmasked attached molecules, and second regions comprising maskedattached molecules, thereby making an array. In one embodiment, themethod further comprises, prior to step c), contacting said modifiedsurface with said second solution. In one embodiment, the method furthercomprises, after step c), contacting said modified surface with saidsecond solution. In one embodiment of the above-described method, saidtemplate of said second solution has been diluted to a concentrationsuch that less than one hundred molecules on average (more preferably,less than 10, still more preferably, less than 1 molecule) of templateare in contact with any one indentation. In one embodiment of theabove-described method, said template of said second solution has beendiluted to a concentration such that less than one hundred molecules(more preferably, less than 10, still more preferably, less than 1molecule) of template are in within any one region. In a preferredembodiment, the template amplified in any one sealed region ishomogeneous (i.e. only one type of template was amplified). In apreferred embodiment, different (diluted) template is introduced into atleast two different sealed regions such that each of said differenttemplate is simultaneously amplified and each of said amplified productis homogeneous. In one embodiment (e.g. where it is desired to havefewer empty wells), said template has been diluted to a concentrationsuch that less than three on average—but more than 1 molecule—oftemplate are in contact with any one indentation (or are within any oneregion). In such a case, the amplified product may not be homogeneous inevery region where there is product.

In one embodiment, said positioning of step d) causes at least a portionof said second solution on said modified surface of said first elementto move off of said surface. In one embodiment, said indentations arewells. In one embodiment, said indentations comprise particles (e.g.beads free or attached to wells). In one embodiment, said first surfaceis substantially flat. In one embodiment, said contacting of step b)results in substantially the entire first surface being contacted withsaid first solution. In one embodiment, said first surface comprisesindentations. In one embodiment, said indentations are evenly spaced. Inone embodiment, said attached molecules after step b) are attached oversubstantially the entire surface at a substantially even density. In oneembodiment, said first element comprises glass. In one embodiment, saidfirst element is a microscope slide. In one embodiment, said firstsurface comprises plastic. In one embodiment, said first surfacecomprises indentations. In one embodiment, said indentations are formedat the time said plastic surface is molded. In one embodiment, saidfirst surface is elastomeric. In one embodiment, said first element is amicrochip. In one embodiment, said first PCR primers consist of forwardPCR primers. In one embodiment, said second PCR primers consist ofreverse PCR primers. In one embodiment, the present inventioncontemplates the array created by the above-described method (as acomposition). In one embodiment, the contacting prior to step c) isunder conditions such that at least a portion of said template isamplified. In one embodiment, the contacting after step c) is underconditions such that at least a portion of said template is amplified.In a preferred embodiment, the template amplified in any one sealedregion is homogeneous (i.e. only one type of template was amplified). Ina preferred embodiment, different (diluted) template is introduced intoat least two different sealed regions such that each of said differenttemplate is simultaneously amplified and each of said amplified productis homogeneous. In one embodiment, the method further comprises the stepd) separating said first element from said second element.

In yet another embodiment, the present invention contemplates a methodfor making an array, comprising: a) providing: i) a first elementcomprising a first surface, said first surface comprising a plurality ofindentations; ii) a second element comprising a second surface; iii) athird element; and iv) a solution comprising molecules selected from thegroup consisting of biomolecules and anchoring molecules; b) contactingsaid first surface with said solution under conditions such that atleast a portion of said molecules attach to at least a portion of saidfirst surface so as to create a modified surface comprising attachedmolecules; c) positioning said second surface on top of said modifiedsurface, so as to create a plurality of first regions defined by saidindentations, said first regions comprising unmasked attached molecules,and second regions comprising masked attached molecules, thereby makingan array; and d) positioning said third element on top of said secondelement, thereby sandwiching said second element between said firstelement and said third element. In one embodiment, said second elementis elastomeric. In one embodiment, said second element is a sheet ofelastomer. In a preferred embodiment, said sheet is a film. In aparticularly preferred embodiment, said molecules are PCR primers, inwhich case it is preferred that said solution comprises nucleic acidtemplate and reagents for PCR (e.g. polymerase, dNTPs, buffer, etc.). Inone embodiment of the above-described method, said template has beendiluted to a concentration such that less than one hundred molecules onaverage (more preferably, less than 10, still more preferably, less than1 molecule) of template are in contact with any one indentation. In oneembodiment of the above-described method, said template of said secondsolution has been diluted to a concentration such that less than onehundred molecules (more preferably, less than 10, still more preferably,less than 1 molecule) of template are within any one region. In oneembodiment (e.g. where it is desired to have fewer empty wells), saidtemplate has been diluted to a concentration such that less than threeon average—but more than 1 molecule—of template are in contact with anyone indentation (or are within any one region).

In one embodiment, the present invention contemplates a method formaking an array, comprising: a) providing: a first element comprising asurface; a second element comprising bottom and top surfaces, saidsecond element further comprising a plurality of channels extending fromsaid bottom to said top surfaces; and a solution comprising moleculesselected from the group consisting of biomolecules and anchoringmolecules; b) contacting the surface of said first element with saidsolution under conditions such that at least a portion of said moleculesattach to at least a portion of said surface (more preferably over theentire surface at a substantially even density) so as to create amodified surface comprising attached molecules; and c) positioning saidbottom surface of said second element on top of said modified surface,so as to create first regions defined by said channels, said firstregions comprising unmasked attached molecules, and second regionscomprising masked attached molecules, thereby making an array.Importantly, the masking the above-described method differs from othermasking processes in that the molecules are attached and thereaftermasked (rather than using the mask to control where the molecules attachin the first place).

The present invention contemplates the array made by the above-describedprocess. The present invention also contemplates the device comprisingsaid first and second elements, as described above.

It is not intended that the present invention be limited to how thesolution is brought into contact with the surface in step b). In oneembodiment, the surface is dipped or immersed in the solution. Inanother embodiment, the solution is poured or pipetted onto the surface.In one embodiment, said positioning of step c) causes at least a portionof said solution (regardless of the manner by which it was introduced)on said surface of said first element to move off (or run off) of saidsurface. It is preferred that at least a portion of said solution remainon said surface after said positioning of step c).

In one embodiment, the method further comprises: d) providing a thirdelement comprising a surface; and e) bringing the surface of said thirdelement into contact with the top surface of said second element,thereby sandwiching said second element between the surfaces of saidfirst and third elements, so as to create a device comprising sealedfirst regions. In one embodiment, the present invention contemplatesthis device, comprising first, second and third elements, said secondelement positioned between said first and third elements, said firstelement comprising masked and unmasked attached molecules.

It is not intended that the present invention be limited to the natureof the first, second or third elements. While these elements may beporous, it is preferred that they are non-porous. Importantly, thenature of each element may be different. In one embodiment, the firstelement is a membrane (e.g. nylon, nitrocellulose, etc.). In oneembodiment, one or more elements are made from one or more polymers. Forexample, in a preferred embodiment, said second element comprises apolymer film.

In a preferred embodiment, said first and third elements are made fromsilicon, quartz or glass. For example, in a preferred embodiment, saidfirst and third elements are glass plates. In a more preferredembodiment, said first and third elements are microscope slides.However, it is not intended that the present invention be limited tolarge surfaces. For example, in one embodiment, said first and thirdelements are microchips or silicon wafers.

It is not intended that the present invention be limited by thetopography of the surfaces. In one embodiment, the surfaces may be flator substantially flat (e.g. flat over 90% of the surface or comprisingless than 10% deviation from flat). In another embodiment, the surfacesmay be curved or raised (or partially curved or partially raised). Inanother embodiment, one or more surfaces may have depressions orprotrusions. The depressions can take the form of wells or pits. Theform of the protrusions can be of any type (e.g. conical or cylindricalshape and a cross-sectional configuration of a polygon, circle, ellipseor a combination thereof) and need not be identical. In an embodimentwith protrusions, the protrusions may be identical in size and shape.

It is not intended that the present invention be limited to elementsthat are homogeneous. For example, crystalline silicon may not be theideal material for biological compatibility. In one embodiment, it maybe desirable to modify the surface with adsorbed surface agents,covalently bonded polymers, or a deposited silicon oxide layer. In oneembodiment, deposition of a metal (e.g. gold) is contemplated. In oneembodiment, deposition of a water-impermeable layer is contemplated onone or more elements. In a preferred embodiment, the impermeable layeris made by a sequence of three plasma enhanced vapor depositions:silicon oxide, silicon nitride, and silicon oxide.

Regardless of the material used, it may be desired in some embodimentsto treat the surface. In one embodiment, the surface comprises an alkynederivatized surface and said biomolecules are azido-labeled. In oneembodiment, the surface is treated with a poly(ethylene glycol) such asa synthetic acridinyl poly(ethylene glycol) (APEG). In one embodiment,the surface is treated with protein such as BSA. In one embodiment, thesurface is pretreated with a “hydrophilicity-enhancing compounds” whichare those compounds or preparations that enhance the hydrophilicity ofthe surface. The definition is functional, rather than structural. Forexample, Rain-X anti-fog is a commercially available reagent containingglycols and siloxanes in ethyl alcohol. However, the fact that itrenders a glass or silicon surface more hydrophilic is more importantthan the reagent's particular formula. In another embodiment, thesurface (or portion thereof) is treated with a “hydrophobic reagents”which are compounds used to make “hydrophobic regions.” It is notintended that the present invention be limited to particular hydrophobicreagents. In one embodiment, the present invention contemplateshydrophobic polymer molecules that can be grafted chemically to thesilicon oxide surface. Such polymer molecules include, but are notlimited to, polydimethylsiloxane. In another embodiment, the presentinvention contemplates the use of silanes to make hydrophobic regions,including but not limited to halogenated silanes and alkylsilanes. Inthis regard, it is not intended that the present invention be limited toparticular silanes; the selection of the silane is only limited in afunctional sense, i.e. that it render the surface hydrophobic. In oneembodiment, the second element (or portion thereof) is dipped in ahydrophobic reagent prior to bringing it into contact with the surfaceof the first element, so as to create hydrophobic regions aroundhydrophilic regions defined by said channels. In one embodiment,n-octadecyltrichlorosilane (OTS) is used as a hydrophobic reagent. Inanother embodiment, octadecyldimethylchlorosilane is employed.

It is not intended that the present invention be limited by the size orshape of the channels of the second element, or the manner in which thechannels of said second element are made. In one embodiment, thechannels are etched. In a preferred embodiment, said second elementcomprises a polymer film treated with a laser to create said channels.While a variety of dimensions are possible, it is generally preferredthat the regions defined by the channels have a width of betweenapproximately 10 and 1000 μm (or greater if desired), and morepreferably between approximately 100 and 500 μm. In a preferredembodiment, said channels are approximately 10-50 microns in diameter(most preferably 20 microns) and are spaced approximately 10-100 micronsapart (most preferably 30 microns).

A variety of thermoplastic or elastomeric materials can be used for thispurpose, such materials typically comprising one or more polymers. Inone embodiment, polymers such as polyvinyl and polyurethane areemployed. In one embodiment, polypropylene is employed. For example,polypropylene can be modified with amino groups by treatment with plasmain a mixture of nitrogen and hydrogen (1:2 V/V). Thereafter, using themethod described above, oligonucleotides may be attached or synthesizedin-situ. In another embodiment, polystyrene (e.g. a polystyrene thinfilm) is employed and amino-modified DNA is attached to the surface byreaction with succinimide ester groups bound to the polystyrenes. In yetanother embodiment, polyethylenimine (PEI) is used. In still anotherembodiment, said polymer comprises a polyimide. In still anotherembodiment, poly-methyl methacrylate (PMMA) is employed. In stillanother embodiment, polydimethylsiloxane (PDMS) material is employed.Mixtures of two or polymers may also be employed. Alternatively, anepoxy-based photosensitive resist (e.g. SU-8) may be used.

It is not intended that the present invention be limited by the natureof the biomolecules used in the above-described method. Biomoleculessuch as enzymes (e.g., polymerases, nucleases, etc.) and nucleic acids(both RNA and DNA) are contemplated. In addition, biomolecules such asproteins (e.g. antibodies) and lipids (e.g. glycosphingolipids) arecontemplated. When nucleic acids are employed, the present inventioncontemplates oligonucleotides, primers (e.g. for PCR), probes (e.g. forRCA) and the like. In some embodiments, nucleic acids having particulardesigns (e.g. hairpins) are desired. In one embodiment of theabove-described method, nucleic acid template is introduced into thedevice. In a preferred embodiment, said template has been diluted to aconcentration such that less than one hundred molecules (morepreferably, less than 10, still more preferably, less than 1 molecule)of template are in contact with any one channel. In a preferredembodiment, said template has been diluted to a concentration such thatless than one hundred molecules on average (more preferably, less than10, still more preferably, less than 1 molecule) of template are withinany one region. In a preferred embodiment, the template amplified in anyone sealed region is homogeneous (i.e. only one type of template wasamplified). In a preferred embodiment, different (diluted) template isintroduced into at least two different sealed regions such that each ofsaid different template is simultaneously amplified and each of saidamplified product is homogeneous. However, in one embodiment (e.g. whereit is desired to have fewer empty channels), said template has beendiluted to a concentration such that less than three on average—but morethan 1 molecule—of template are in contact with any one channel (or arewithin any one region). In such a case, the amplified product within aregion may not be homogeneous.

A variety of anchoring molecules can be used, including chemicalmoieties (epoxide groups, ester groups, amino groups, etc.) andbiomolecules that function as anchoring molecules (e.g. biotin, avidin,etc.). Molecules function as anchoring molecules when they permit theattachment and immobilization of other molecules.

In another embodiment, the present invention contemplates a method formaking an array, comprising: a) providing: a first element comprising asurface; a second element comprising bottom and top surfaces, saidsecond element further comprising a plurality of channels extending fromsaid bottom to said top surfaces; and a solution comprising forward andreverse PCR primers; b) positioning said bottom surface of said secondelement on top of said surface of said first element, so as to createunmasked regions defined by said channels, and masked regions; and c)contacting the surface of said first element with said solution underconditions such that at least a portion of said PCR primers attach to atleast a portion of said surface so as to create a modified surfacecomprising attached molecules, said attached molecules positioned insaid unmasked regions, thereby making an array. In one embodiment, saidmasked regions are free of attached molecules. In one embodiment, saidcontacting of step c) comprises introducing said solution into saidchannels. In one embodiment, said solution further comprises template,polymerase and dNTPs (or a second solution containing these componentsis introduced), and at least a portion of said PCR primers amplify atleast a portion of said template. In a preferred embodiment, saidtemplate has been diluted to a concentration such that less than onehundred molecules on average (more preferably, less than 10, still morepreferably, less than 1 molecule) of template are in contact with anyone channel (or within any one unmasked region). In a preferredembodiment, the template amplified in any one unmasked region ishomogeneous (i.e. only one type of template was amplified). In apreferred embodiment, different (diluted) template is introduced into atleast two different unmasked regions such that each of said differenttemplate is simultaneously amplified and each of said amplified productis homogeneous. However, in one embodiment (e.g. where it is desired tohave fewer empty channels), said template has been diluted to aconcentration such that less than three on average—but more than 1molecule—of template are in contact with any one channels (or are withinany one region). In such a case, the amplified product may not behomogeneous in every instance.

In one embodiment, the method further comprises: providing a thirdelement comprising a surface; and bringing the surface of said thirdelement into contact with the top surface of said second element,thereby sandwiching said second element between the surfaces of saidfirst and third elements, so as to create a device comprising sealedunmasked regions. In one embodiment, the present invention contemplatesthis device, comprising first, second and third elements, said secondelement positioned between said first and third elements, said firstelement comprising unmasked attached molecules.

Again; it is not intended that the present invention be limited by thenature or topography of the elements. In one embodiment, said firstelement is substantially flat, the bottom surface of said second elementis substantially flat, and the surface of said third element issubstantially flat. On the other hand, these surfaces may be curved orraised (or partially curved or raised), smooth or rough, with or withoutdepressions or protrusions. In one embodiment, said first and thirdelements comprise glass (e.g. glass plates, microscope slides, etc.). Inone embodiment, said first and third elements are microchips.

It is not intended that the present invention be limited by thedimensions or spacing of the channels. However, in one embodiment saidchannels of said second element are evenly spaced.

It is not intended that the present invention be limited to the extent asurface is covered with attached molecules. However, in one embodimentsaid attached molecules after step b) are attached over substantiallythe entire exposed surface at a substantially even density.

In a preferred embodiment, the present invention contemplates a methodfor making an array, comprising: a) providing: first and third elementseach comprising a substantially flat surface; a second elementcomprising substantially flat bottom and top surfaces, said secondelement further comprising a plurality of substantially evenly spacedchannels extending from said bottom to said top surfaces; and a solutioncomprising molecules selected from the group of biomolecules andanchoring molecules; b) contacting the substantially flat surface ofsaid first element with said solution under conditions such that atleast a portion of said molecules attach over substantially the entiresurface at a substantially even density so as to create a modifiedsurface comprising attached molecules; c) positioning said bottomsurface of said second element on top of said modified surface, so as tocreate first regions defined by said channels, said first regionscomprising unmasked attached molecules, and second regions comprisingmasked attached molecules, thereby making an array; and d) bringing thesurface of said third element into contact with the top surface of saidsecond element, thereby sandwiching said second element between thesurfaces of said first and third elements, so as to create a devicecomprising sealed first regions. Again, the present inventioncontemplates the device created by the method described above, i.e. adevice comprising first, second and third elements, said second elementpositioned between said first and third elements, said first elementcomprising attached molecules.

Again, it is not intended that the present invention be limited by thenature or topography of the elements. The surfaces may be smooth orrough, with or without depressions or protrusions. In one embodiment,said first and third elements comprise glass (e.g. glass plates,microscope slides, etc.). In one embodiment, said first and thirdelements are microchips. In one embodiment said second element comprisesa polymer film (e.g. polyimide or other suitable polymer as discussedpreviously) treated (e.g. with a laser or by another process) to createsaid channels.

Again, it is not intended that the present invention be limited by thedimensions or spacing of the channels. However, in one embodiment saidchannels of said second element are evenly spaced. In one embodiment,said channels are approximately 10-50 microns (preferably 20 microns) indiameter and are spaced approximately 10-100 microns (preferably 30microns) apart.

Again, it is not intended that the present invention be limited to theextent a surface is covered with attached molecules. However, in oneembodiment said attached molecules after step b) are attached oversubstantially the entire exposed surface at a substantially evendensity.

Again, it is not intended that the present invention be limited by thenature of the anchoring molecules or biomolecules. Where said moleculesare biomolecules, the preferred biomolecule comprises nucleic acidmolecules. Particularly useful nucleic acid molecules include (but arenot limited to) probes for rolling circle amplification (RCA), primersfor PCR, and oligonucleotides for sequencing by synthesis. With regardto the latter, a preferred oligonucleotide comprises one or morehairpins. In one embodiment, template is introduced into the device(e.g. for amplification). In a preferred embodiment, said template hasbeen diluted to a concentration such that less than one hundredmolecules on average (more preferably, less than 10, still morepreferably, less than 1 molecule) of template are in contact with anyone channel (or within any one region). In a preferred embodiment, thetemplate amplified in any one sealed region is homogeneous (i.e. onlyone type of template was amplified). In a preferred embodiment,different (diluted) template is introduced into at least two differentsealed regions such that each of said different template issimultaneously amplified and each of said amplified product ishomogeneous. However, in one embodiment (e.g. where it is desired tohave fewer empty channels), said template has been diluted to aconcentration such that less than three on average—but more than 1molecule—of template are in contact with any one channel (or within anyone region). In such a case, the amplified product may not behomogeneous in every instance.

The arrays described above are useful for a variety of tasks, includingbut not limited to amplification (including amplification done inadvance of sequencing). In one embodiment, the present inventioncontemplates a method of amplifying nucleic acid template, comprising:a) providing: first and third elements, each comprising a surface; asecond element comprising bottom and top surfaces, said second elementfurther comprising a plurality of channels extending from said bottom tosaid top surfaces; a first solution comprising primers; and a secondsolution comprising template, polymerase and dNTPs; b) contacting atleast a portion of said surface of said first element with said firstsolution under conditions such that at least a portion of said primersattach so as to create a modified surface comprising attached primers;c) positioning said bottom surface of said second element on top of saidmodified surface, so as to create first regions defined by saidchannels, said first regions comprising unmasked attached primers, andsecond regions comprising masked attached primers, thereby making anarray; d) introducing said second solution into said first regions; e)bringing the surface of said third element into contact with the topsurface of said second element, thereby sandwiching said second elementbetween the surfaces of said first and third elements, so as to createsealed first regions of a device; and f) treating said sealed firstregions under conditions such that said template is amplified. In oneembodiment, said treating of step (f) comprises thermally cycling thedevice of step (e). In one embodiment, said first solution comprisesforward primers for PCR. In one embodiment, said first solution furthercomprises reverse primers for PCR. In a preferred embodiment, saidtemplate has been diluted to a concentration such that less than onehundred molecules on average (more preferably, less than 10, still morepreferably, less than 1 molecule) of template are in contact with anyone channel (or within any one sealed region). In a preferredembodiment, the template amplified in any one sealed region ishomogeneous (i.e. only one type of template was amplified). In apreferred embodiment, different (diluted) template is introduced into atleast two different sealed regions such that each of said differenttemplate is simultaneously amplified and each of said amplified productis homogeneous. However, in one embodiment (e.g. where it is desired tohave fewer empty regions), said template has been diluted to aconcentration such that less than three on average—but more than 1molecule—of template are within any one region. In such a case, theamplified product may not be homogeneous in every instance.

In one embodiment, said surfaces of said first and third elements aresubstantially flat. In one embodiment, said first and third elementscomprise glass. In one embodiment, said first and second elements aremicroscope slides. In one embodiment, said first and second elements aremicrochips. In one embodiment, said third element comprises a polymerfilm treated with a laser to create said channels. In one embodiment,said channels are approximately 20 microns in diameter and are spacedapproximately 30 microns apart. In one embodiment, the present inventioncontemplates the device (as a composition) with the elements assembledas described above.

In yet another embodiment, the present invention contemplates a methodof amplifying nucleic acid template, comprising: a) providing: i) firstand third elements, each comprising a surface; ii) a second elementcomprising bottom and top surfaces, said second element furthercomprising a plurality of channels extending from said bottom to saidtop surfaces; iii) a first solution comprising primers; and iv) a secondsolution comprising template, polymerase and dNTPs; b) positioning saidbottom surface of said second element on top of said surface of saidfirst element, so as to create unmasked regions defined by saidchannels, and masked regions; c) contacting at least a portion of saidsurface of said first element with said first solution under conditionssuch that at least a portion of said primers attach in said unmaskedregions so as to create a modified surface comprising attached primers;d) introducing said second solution into said unmasked regions; e)bringing the surface of said third element into contact with the topsurface of said second element, thereby sandwiching said second elementbetween the surfaces of said first and third elements, so as to createsealed unmasked regions of a device; and f) treating said sealed firstregions under conditions such that said template is amplified. In oneembodiment, said treating of step (f) comprises thermally cycling thedevice of step (e). In one embodiment, said first solution comprisesforward primers for PCR. In one embodiment, said first solution furthercomprises reverse primers for PCR. In a preferred embodiment, saidtemplate has been diluted to a concentration such that less than onehundred molecules on average (more preferably, less than 10, still morepreferably, less than 1 molecule) of template are in contact with anyone channel (or within any one sealed unmasked region). In a preferredembodiment, the template amplified in any one sealed unmasked region ishomogeneous (i.e. only one type of template was amplified). In apreferred embodiment, different (diluted) template is introduced into atleast two different sealed unmasked regions such that each of saiddifferent template is simultaneously amplified and each of saidamplified product is homogeneous. However, in one embodiment (e.g. whereit is desired to have fewer empty unmasked regions), said template hasbeen diluted to a concentration such that less than three on average—butmore than 1 molecule—of template are within any one unmasked region).

In one embodiment, said surfaces of said first and third elements aresubstantially flat. In one embodiment, said first and third elementscomprise glass. In one embodiment, said first and second elements aremicroscope slides. In one embodiment, said first and second elements aremicrochips. In one embodiment, said third element comprises a polymerfilm treated with a laser to create said channels. In one embodiment,said channels are approximately 20 microns in diameter and are spacedapproximately 30 microns apart. In one embodiment, the present inventioncontemplates the device with the elements assembled as described above.

In yet another embodiment, the present invention contemplates a methodof amplifying nucleic acid template, comprising: a) providing: i) firstand third elements, each comprising a surface; ii) a second elementcomprising bottom and top surfaces, said second element furthercomprising a plurality of channels extending from said bottom to saidtop surfaces; iii) a first solution comprising a plurality of forwardPCR primers; iv) a second solution comprising a plurality of reverse PCRprimers; and v) a third solution comprising template, polymerase anddNTPs; b) contacting at least a portion of said surface of said firstelement with said first solution under conditions such that at least aportion of said forward primers attach so as to create a first modifiedsurface comprising attached forward primers; c) contacting at least aportion of said surface of said third element with said second solutionunder conditions such that at least a portion of said reverse primersattach so as to create a second modified surface comprising attachedreverse primers; d) positioning said bottom surface of said secondelement on top of said first modified surface, so as to create firstregions defined by said channels, said first regions comprising unmaskedattached forward primers, and second regions comprising masked attachedforward primers; e) introducing said second solution into said firstregions; f) bringing said second modified surface into contact with thetop surface of said second element, thereby sandwiching said secondelement between the modified surfaces of said first and third elements,so as to create sealed first regions of a device, said sealed firstregions comprising unmasked attached forward and reverse primers; and g)treating said sealed first regions under conditions such that saidtemplate is amplified. In a preferred embodiment, the template amplifiedin any one sealed region is homogeneous (i.e. only one type of templatewas amplified). In a preferred embodiment, different (diluted) templateis introduced into at least two different sealed regions such that eachof said different template is simultaneously amplified and each of saidamplified product is homogeneous. However, in one embodiment (e.g. whereit is desired to have fewer empty channels), said template has beendiluted to a concentration such that less than three on average—but morethan 1 molecule—of template are in contact with any one channel (or arewithin any one region).

In one embodiment, said treating of step (g) comprises thermally cyclingthe device of step (f). In one embodiment, the present inventioncontemplates the device with the elements assembled as described above.In one embodiment, said first and third elements comprise glass. In oneembodiment, said first and third elements are microscope slides. In oneembodiment, said first and third elements are microchips. In oneembodiment, said second element comprises a polymer film. In oneembodiment, said polymer film is treated with a laser to create saidchannels.

In one embodiment, both the forward and reverse primers are attached tothe same surface in the same or different concentrations. In thisembodiment, amplification causes the generation of DNA strands attachedto the surface by either the forward or reverse primer. In oneembodiment, a separate sequencing primer is then used to determine thesequence of the forward single strands; then, a re-priming step with adifferent sequencing primer is used to determine the sequence of thereverse single strands. In this manner, one can get sequence informationfrom both ends.

To maximize the efficiency of generating both forward and reversestrands on a solid surface, it is useful to have the amplification occurin two phases under different conditions. For high efficiency ofsolid-surface PCR, it is desirable to have only one of the two primerson the surface with the other primer in solution; thus, amplicons areforced to use the surface-bound primers and become attached. Such solidsurface could be a surface of a chip or a surface of a microsphere orbead. The efficiency of the reaction is helped with the use of alimiting concentration of the bound primer in solution. In oneembodiment, the supply of solution-based primers is regulated based onthe reaction and/or structural conditions (i.e. melting temperature,hairpins, cleavable blocker, etc). For example, if one inhibits theannealing of solution-based reverse primers, but allow for the annealingof an abundance of solution-based forward primers, then we can drive theamplification of fragments that are only attached through the reverseprimers on the solid surface (only reverse primers that are availableare on the surface). However, if required, a small quantity of activereverse primer could added to improve efficiency. As this reactionprogresses, the solution-based forward primers will be exhausted. Duringa second phase of the reaction, the solution-based (inactive) reverseprimers will be activated by some means (change in temperature, removalof blocking group, etc) and amplification occurs with strands beingattached through the forward primers affixed to the solid surface.Specific examples of primer activation could include, by are not limitedto, a photocleavable blocking group or by changing the PCR annealingtemperature allowing initially dormant reverse primers designed with orwithout hairpins to become active in the second phase of a PCR reaction.

In yet another embodiment, the present invention contemplates a methodof amplifying nucleic acid template, comprising: a) providing i) apopulation of nucleic acid template molecules and ii) a plurality ofregions, said regions defined by a plurality of indentations in a firstsurface, said first surface in contact with a second surface, whereineach of said regions comprises one or more PCR primers; and b)amplifying, in said regions, at least a portion of said population ofnucleic acid template molecules. In a preferred embodiment, the templateamplified in any one sealed region is homogeneous (i.e. only one type oftemplate was amplified). In a preferred embodiment, different (diluted)template is introduced into at least two different sealed regions suchthat each of said different template is simultaneously amplified andeach of said amplified product is homogeneous. However, in oneembodiment (e.g. where it is desired to have fewer empty regions), saidtemplate has been diluted to a concentration such that less than threeon average—but more than 1 molecule—of template are within any oneregion. In such a case, the amplified product may not be homogeneous butmay be substantially homogeneous (i.e. the product of not more thanthree different templates).

In one embodiment, said PCR primers are attached to either said firstsurface or said second surface. In one embodiment, said amplifying instep b) is performed in a solution comprising said template molecules,one or more polymerases, and all four dNTPs. In one embodiment, saidsolution further comprises unattached PCR primers. In one embodiment,the concentration of said template molecules in said solution is suchthat there is less than one template molecule on average per region. Inone embodiment, the concentration of said template molecules in saidsolution is such that there is greater than one template molecule onaverage per region. In one embodiment, the concentration of saidtemplate molecules in said solution is such that there are greater thantwo template molecules on average per region. In one embodiment, theconcentration of said template molecules in said solution is such thatthere are not greater than three template molecules on average perregion.

In yet another embodiment, the present invention contemplates a methodof amplifying nucleic acid template, comprising: a) providing apopulation of nucleic acid template molecules and a device comprising afirst surface comprising attached PCR primers in contact with a secondsurface comprising attached PCR primers, either said first surface orsaid second surface comprising a plurality of indentations, saidindentations defining a plurality of regions, said regions comprisingunmasked PCR primers; and b) amplifying at least a portion of saidpopulation of nucleic acid template molecules with said unmasked PCRprimers in one or more of said regions. In one embodiment, said attachedPCR primers of said first and second surfaces that are outside saidregions are masked by said contact. In one embodiment, said amplifyingin step b) is performed in a solution comprising said templatemolecules, one or more polymerases, and all four dNTPs. In oneembodiment, said solution further comprises unattached PCR primers. Inone embodiment, the concentration of said template molecules in saidsolution is such that there is less than one template molecule onaverage per region. In one embodiment, the concentration of saidtemplate molecules in said solution is such that there is greater thanone template molecule on average per region. In one embodiment, theconcentration of said template molecules in said solution is such thatthere are greater than two template molecules on average per region. Inone embodiment, the concentration of said template molecules in saidsolution is such that there are not greater than three templatemolecules on average per region.

In one embodiment, the present invention contemplates a method,comprising: a) providing: I) a first element comprising a surface; ii) asecond element comprising bottom and top surfaces, said bottom surfacecomprising a plurality of indentations; iii) a first solution comprisinga plurality of first PCR primers; iv) a second solution comprising aplurality of second PCR primers; and v) a third solution comprisingtemplate at a known concentration; b) contacting the surface of saidfirst element with said first solution under conditions such that atleast a portion of said first PCR primers attach to at least a portionof said surface so as to create a first modified surface comprisingattached molecules; c) contacting the bottom surface of said secondelement with said second solution under conditions such that at least aportion of said second PCR primers attach to at least a portion of saidbottom surface so as to create a second modified surface comprisingattached molecules; and positioning said second modified surface on topof said first modified surface, so as to create a plurality of firstregions defined by said indentations, said first regions comprisingunmasked attached molecules, and second regions comprising maskedattached molecules, thereby making an array. In one embodiment, themethod further comprises introducing said third solution to said arrayunder conditions such that at least a portion of said template isamplified, wherein the known concentration is such that less than onetemplate molecule is present on average in each of said plurality offirst regions. In one embodiment, the method further comprisesintroducing said third solution to said array under conditions such thatat least a portion of said template is amplified, wherein the knownconcentration is such that greater than one template molecule is presenton average in each of said plurality of first regions. In oneembodiment, the method further comprises introducing said third solutionto said array under conditions such that at least a portion of saidtemplate is amplified, wherein the known concentration is such thatgreater than two template molecules are present on average in each ofsaid plurality of first regions. In one embodiment, said introducing ofsaid third solution is prior to step d). In one embodiment, said thirdsolution further comprises at least one polymerase and all four dNTPs.In one embodiment, said third solution further comprises PCR primers.

In one embodiment, the method further comprises the step e) separatingsaid first element from said second element.

DESCRIPTION OF THE FIGURES

FIG. 1 shows one embodiment of a device for amplification on a surface.FIG. 1A shows the three elements of the device (a bottom piece, a toppiece, and a middle piece) not yet combined. A solution (typicallycontaining biomolecules, such as nucleic acid template and reagents forPCR) is shown positioned on the bottom piece such that fluid has enteredthe wells. FIG. 1B shows the assembled device wherein the middle piece(in this case, an elastomeric sheet) acts as a seal, trapping fluid inthe wells. Where primers have been attached to the surface of the bottompiece prior to assembly, the middle piece will mask the primers on thesurface interface, but leave the primers attached to the wells unmaskedand functional for amplification. Because of the excess fluid, a portionof the solution positioned on the bottom surface typically runs off thebottom surface when the middle piece is applied and the device isassembled. After the device is used (e.g. nucleic acid within the wellsis amplified by thermocycling the device, or portion thereof), it can betaken apart, resulting in the three separated elements.

FIG. 2 shows another embodiment, wherein the device is characterized bychannels created by top and bottom pieces (without indentations)separated by a third piece (which can be, in one embodiment, aperforated polymeric gasket). FIG. 2A shows the assembled device. Whereprimers have been attached to the surface of the bottom piece prior toassembly (as shown in 2A), the middle piece will mask the primers at thepoint of contact, but leave the primers in the channels unmasked andfunctional for amplification. After the device is used (e.g. nucleicacid template within the channels is amplified by thermocycling thedevice, or portion thereof), it can be taken apart (as shown in 2B),resulting in a surface comprising discrete regions comprising amplifiedproduct, i.e. an array.

FIG. 3 shows one embodiment of the method of utilizing one embodiment ofthe device of the present invention to create amplified product.

FIG. 4 shows a Poisson probability distribution with varying averagemolecule densities per well (m).

FIG. 5 shows a perforated gasket with 30 micron holes at 50 micronspacing through a 25 micron thick polyimide film.

FIG. 6 is a scanned fluorescent image from single base extensionreactions on DNA templates bound to the surface of a glass slide using apatterned (“MIT”) prototype chip. The integrated chip was formed from aglass slide and a molded PDMS piece that had 40 micron holes at about a200 micron spacing. The scanner resolution was 5 microns.

FIG. 7 is a fluorescent seamier image of a glass slide showing PCRamplification of primers bound to a slide.

DESCRIPTION OF THE INVENTION

The present invention relates to methods and devices for amplifyingnucleic acid, and, in particular, amplifying so as to generate productson a surface without the use of emulsions. In a preferred embodiment, aplurality of groups of amplified product are generated on the surface,each group positioned in different (typically predetermined) locationson said surface so as to create an array. In one embodiment, each groupis homogeneous. In one embodiment, each group consists of amplifiedproduct of a single nucleic acid template. In one embodiment, the methodcomprises performing limiting dilution PCR within closed compartments(e.g. sealed regions) created by two surfaces coming together.Performing a limiting dilution PCR on a surface (e.g. surface of aslide, chip, etc.) rather than in emulsion allows for a simpler and lesscumbersome approach. In one embodiment, the present inventioncontemplates that the device a) isolates each region (e.g. reactionsite) from one another and b) contains them for thermal cycling. In oneembodiment, the device is disposable.

FIG. 1A shows an embodiment wherein the top surface (10) of the bottompiece (11) has a plurality of indentations (9); however, in otherembodiments, the bottom surface (12) of the top piece (13) hasindentations, or both pieces have indentations. FIG. 1B shows anassembled three piece embodiment (17); however, in some embodiments, themiddle piece (14) is eliminated and the bottom surface (12) of the toppiece (13) is simply brought into contact with the top surface (10 ofthe bottom piece (11). This will also cause a portion of the solution(15) to run off the bottom piece (11), although a portion (16) willremain in the indentations (9), i.e. they will be fluid-filled (althoughthey need not be completely filled). The bottom surface (12) of the toppiece (13) in this embodiment may or may not have biomolecules (e.g.primers) attached thereto (not shown). Whether the middle piece (14) isused or not, biomolecules on the contact points (8) (i.e. at theinterface between the surfaces) will be masked, while biomolecules (e.g.primers) within the indentations (9) will be unmasked and functional.Template in the solution (15) can be diluted to a concentration wherebyeach indentation (9) on average contains between 1 and 100 molecules,and more preferably, 1 and 3 molecules of template at the point thedevice is assembled (or, if desired, less than 1 molecule on average).

FIG. 2A shows an embodiment of an assembled three-piece device (20)wherein the primers (21) are only on the top surface (22) of the bottompiece (23). The middle piece (18) masks the primers where it contactsthe top surface (22). Once the device is thermocycled and taken apart(FIG. 2B), the result is amplified product (24) in discrete regions (25)on one surface, i.e. one array (26). However, the present inventioncontemplates embodiments wherein both surfaces comprise primers; forexample, the bottom surface (27) of the top piece (28) can also compriseprimers (not shown) and the result is amplified product on bothsurfaces, i.e. two arrays (one being the mirror image of the other).Such arrays can be used for standard biological assays (e.g.hybridization, sequencing, etc.).

FIG. 2A shows a molecule of template (29) suspended in solution. Thesolution can be diluted to maximize the chance of having one (or a few)starting molecules in each chamber. Template (29) in the solution can bediluted to a concentration whereby each channel (30) on average containsbetween 1 and 100 molecules, and more preferably, 1 and 3 molecules oftemplate at the point the device is assembled. Or, if desired, thesolution can be diluted to maximize the chance of have no more than onestarting molecules in each chamber. For example, template (29) insolution can be diluted to a concentration whereby each channel (30) onaverage contains less than 1 molecule).

While FIG. 2 has been illustrated with reference to primers, otherbiomolecules are contemplated. For example, enzymes might be attachedthe bottom surface (or both surfaces) and masked by the middle piece soas to create reaction channels. Substrate could be processed in thechannels and the result captured in an array format. Similarly,antibodies, receptors and the like can be similarly arrayed.

FIG. 3 shows one embodiment of the method of utilizing one embodiment ofthe device of the present invention to create amplified product. Step 1comprises coating a surface (31) with a biomolecule to create attachedbiomolecules (32) (e.g. attached primer(s)). Step 2 comprises a) maskinga portion of the attached biomolecules (32) using a middle piece (orpieces) at the interface (19) of the coated surface (31) and the middlepiece (33), b) leaving a portion of the attached biomolecules (32)unmasked in discrete regions (34) (e.g. channels), and c) introducing asolution (35) comprising unattached biomolecules (e.g. template,polymerase, etc.). Step 3 comprises sealing the channels (34) with a toppiece (36) to create sealed regions (37) (e.g. sealed compartments,sealed chambers, etc.) wherein the unmasked attached biomolecules (32)are functional. Step 4 comprises initiating a reaction (e.g. PCR bythermocycling the assembled device (38)) so as to create product (39)(e.g. amplified product from PCR). Step 5 involves taking the assembleddevice (38) apart, thereby removing the top piece (36) and the middlepiece or pieces (33) so as to provide a surface (31) with product (39)in a plurality of discrete regions (40). Step 6 (optional) compriseswashing to ensure the removal of all unattached biomolecules (41).

DESCRIPTION OF PREFERRED EMBODIMENTS

In one embodiment, the present invention contemplates using a limitingdilution technique to provide conditions such that PCR products may begenerated from a single molecule (i.e., for example, a DNA template ornucleic acid fragment). By performing the limiting dilution techniqueprior to the PCR where less than one template molecule on averagecontacts the indentations (e.g. wells) or channels, most of the wells orchannels will contain either a single molecule of DNA or no DNA.Relatively few wells or channels will contain multiple templates.Clearly, a less dilute solution will result in a greater number oftemplate molecules per well (and fewer empty wells). Although it is notnecessary to understand the mechanism of an invention, it is believedthat the Poisson distribution governs the distribution of fragments inwells:

$\begin{matrix}{{{P(a)} = {^{- m}( \frac{m^{a}}{a!} )}},} & {(1).}\end{matrix}$

where P(α) is the probability of a well having some integer number ofmolecules (a) based upon a per well average number of availablemolecules (m). In one embodiment, (m) is equivalent to a specificdilution level. Performing PCR on a set of highly dilute wells will thengenerate some number of wells with copies of identical molecules and afew other wells with different templates. Indeed, the present inventioncontemplates in one embodiment methods and devices wherein differenttemplate are placed in the same indentation or channel.

For example, a representative Poisson distribution can be generatedusing a number of values of (m). Changing (m) means that thedistribution of the number of molecules per region changes. See FIG. 4.This illustration shows that in order to minimize the number of wellswith different templates, a dilution providing an average of 0.25molecules per well (e.g., m=0.25), generates a library where 2.6% of thewells contain different templates. Consequently, approximately 20% ofthe wells contain single molecules and approximately 78% of the wellscontain no molecules.

In one embodiment, the present invention contemplates providing wellswith either a single template or several different templates. Oneadvantage of this embodiment is that a dilution level (m) may be chosenthat maximizes the number templates that will be amplified, but does notuse too many templates to provide useful results (i.e., for example,more than four). In a preferred embodiment, each well has between 1 and3 templates on average. To maximize the number of single moleculeregions, in one embodiment, the optimal dilution is chosen as m=1 (linewith diamonds in FIG. 4).

As just described, limiting dilution may result in any particular welli) being empty; ii) consisting of a single DNA template; or iii)comprising two or more different DNA templates. In the case of a singleDNA template, universal primers may be employed to amplify the singlemolecule to saturation. Although it is not necessary to understand themechanism of an invention, it is believed that PCR under limitingdilution conditions will start more slowly than a standard PCR assaysince it will be more difficult for the polymerase to “find” the singleDNA template. It is further believed that it may take up to 60 cycles ina thermal cycler to reach saturation. When two or more differenttemplates are within the same well (i.e., multiplexed) proper PCRconditions are not as predictable. In one embodiment, the presentinvention contemplates methods wherein a first DNA template is amplifiedfaster than a second DNA template in a multiplexed amplificationconfiguration.

EXPERIMENTAL

Some of the embodiments described above are illustrated below withexperimental examples. Of course, such examples are not meant to limitthe scope of the claims in any manner.

Example 1

In this example, the generation of prototype gaskets (for use as amiddle piece in one embodiment of the device, see FIG. 2) by laserdrilling holes through polymer films is described. Both polyimide andpolycarbonate sheets have been employed and thicknesses of both 13microns and 50 microns have been tested. FIG. 5 shows one embodiment ofa prototype gasket fabricated from 25 micron thick polyimide film (42)with 30 micron holes (43) drilled at 50 micron spacing several hundredat a time by an Excimer laser system.

Example 2

In this example, a prototype molded piece of polydimethylsiloxane (PDMS)material was employed which had 40 micron wells as small reactionchambers. The piece was molded using a micromachined silicon wafer asthe negative mold for the PDMS. When cured, PDMS is a flexible polymerwhich is frequently used to create microchannel fluidic systems for avariety of applications. The PDMS piece had a pattern of 40 micron holesat 200 micron spacing. It was clamped against an epoxide activatedmicroarray slide (Corning, Corning, N.Y.) which was covered with asolution of oligonucleotide templates with an amine group on the 5′ endand a hairpin on the 3′ end (used for extension priming). This entireassembly was left overnight for the amine groups to bond with the epoxygroups. The assembly was then taken apart and a single base extensionreaction with labeled nucleotide and Klenow fragment polymerase wasperformed on the slide. The slide was scanned in a General ScanningScanArray 4000 scanner (5 micron resolution) and is shown in FIG. 6A.The spots (44) on the chip produced about 100 times the fluorescentsignal as the areas which were masked by the PDMS piece. A highermagnification (6B) of a region is also shown.

Example 3

In this example, solid phase PCR is described. The feasibility of deviceshown in FIG. 2 was demonstrated by performing the reaction in asomewhat larger enclosed chamber on a glass chip. The Corning epoxideglass surface was spotted in two places with a forward PCR primerattached through an amino group at the 5′ end. The primer was designedto contain a linker that would allow the primer to be elevated away fromthe glass surface. The linker structure contained a 5′ amino group andtwo 18 atom hexa-ethyleneglycol linkers. The reverse PCR primer was freein solution and had a fluorescent label at the 5′ end. Labeled ten-merpoly-T oligonucleotides were also spotted onto the glass surface outsidethe reaction chamber with the same linker attachment groups. Thereaction chamber was formed around the bound forward primers by a one cmsquare adhesive gasket and a thin plastic barrier. The PCR reactionsolution was added inside the gasket and then sealed, with the barrier.The slide (45) was sandwiched between metal plates and placed onto aBio-Rad PTC-200 thermal cycler with a heated lid. The entire sandwichwas cycled through three temperatures of 95, 50, and 72° C. for 30cycles. Following the completion of thermal cycling, the sandwich wasdisassembled and the slide was washed for one hour in PCR buffer andwater. It was then dried and imaged on a ScanArray 4000 microarrayscanner. The bound, labeled amplicons, FIG. 7, show the PCR reaction wassuccessful.

Example 4

In this example, two surfaces (e.g. glass slides) are used to sandwich amiddle piece (e.g. perforated polymeric gasket) and create many smallcylindrical compartments for housing PCR reactions (FIG. 3). Theperforated film between the slides has potentially millions of holes atclose spacing and is about 50 microns thick. The surface of at least oneof the slides which faces the gasket has at least one of the PCR primers(forward or reverse, or both) attached to it (FIG. 3, step (1)). Thereaction cocktail of templates, primers, dNTPs, polymerase and buffer isadded, the gasket is placed over the coated surface, and then the secondsurface is placed on top of the entire assembly (FIG. 3 steps (2 and3)). During assembly, any extra fluid is allowed to escape out the sidesof the sandwich and the entire assembly is clamped together. The gasketmay be treated on one or two sides with an adhesive material to eithereliminate the clamping or help to facilitate sealing. The entire securedassembly is then subjected to thermal cycling and PCR amplificationoccurs in each of the chambers created by the gasket holes and the twoslides (FIG. 3 step (4)). At the end of the amplification, a number ofamplicons are formed and some are attached to the surface throughextension of the primers which were attached to the surface prior toamplification. No amplicons are attached to the primers which are maskedby the gasket material (not in the chambers). Thus, when the sandwich isdisassembled (FIG. 3 step (5)), the result will be a surface with aplurality of discrete groups comprising DNA amplicon attached in anarray of the same pattern as the holes in the gasket which were used(FIG. 3 step (6)). The top glass slide and gasket may also be made fromone piece which both provides the chambers for PCR and creates a sealagainst the bottom slide.

Example 5

As noted above, in one embodiment, both the forward and reverse primersare attached to the same surface. In this embodiment, amplificationcauses the generation of forward single strands and reverse singlestrands. In one embodiment, a separate sequencing primer is then used todetermine the sequence of the forward single strands; then, a re-primingstep with a different sequencing primer is used to determine thesequence of the reverse single strands. In this manner, one can getsequence information from both ends.

In some embodiments, the present invention contemplates, activatable(temporarily inactive) oligonucleotide primers are employed. Forexample, when the first sequencing primer is used, the second sequencingprimer is inactive. In this example, an activatable primer has aphosphorylated 3′-terminal phosphate that prevents the primers fromthemselves being extended. To activate the primer, the phosphate groupis removed by treatment with a phosphatase enzyme, thereby “activating”the primer. One suitable phosphatase enzyme is alkaline phosphatase.

1. A method of amplifying and sequencing nucleic acid, comprising: a)providing i) a population of different nucleic acid template molecules,ii) a plurality of first and second single stranded oligonucleotidesimmobilized on a glass surface, iii) amplification reagents, iv)sequencing reagents, and v) a plurality of first and second sequencingprimers; b) hybridizing at least a portion of said population of nucleicacid template molecules to said plurality of first oligonucleotidesimmobilized on said glass surface; c) amplifying said nucleic acidtemplate molecules so as to create a plurality of forward and reversesingle stranded strands; d) sequencing said forward oligonucleotidestrands with said first sequencing primers; and e) sequencing saidreverse oligonucleotide strands with said second sequencing primers. 2.The method of claim 1, wherein said amplification reagents comprisepolymerase and dNTPs.
 3. The method of claim 1, wherein said sequencingreagents comprise reagents for sequencing by synthesis.
 4. The method ofclaim 1, wherein said plurality of first and second single strandedoligonucleotides are immobilized to said glass surface through a linker.5. The method of claim 4, wherein said linker elevates saidoligonucleotides away from said glass surface.
 6. The method of claim 1,wherein when the first sequencing primers are used in step d), saidsecond sequencing primers are not active.
 7. A method of amplifying andsequencing nucleic acid, comprising: a) providing i) a population ofdifferent nucleic acid template molecules, ii) a plurality of first andsecond single stranded oligonucleotides immobilized on a glass surface,iii) amplification reagents, iv) sequencing reagents, and v) a pluralityof first and second sequencing primers; b) hybridizing at least aportion of said population of nucleic acid template molecules to saidplurality of first oligonucleotides immobilized on said glass surface;c) amplifying said nucleic acid template molecules so as to create aplurality of forward strands; d) sequencing said forward oligonucleotidestrands with said first sequencing primers; e) amplifying said nucleicacid template molecules so as to create a plurality of reverse singlestranded strands; and f) sequencing said reverse oligonucleotide strandswith said second sequencing primers.
 8. The method of claim 7, whereinsaid amplification reagents comprise polymerase and dNTPs.
 9. The methodof claim 7, wherein said sequencing reagents comprise reagents forsequencing by synthesis.
 10. The method of claim 7, wherein saidplurality of first and second single stranded oligonucleotides areimmobilized to said glass surface through a linker.
 11. The method ofclaim 10, wherein said linker elevates said oligonucleotides away fromsaid glass surface.
 12. The method of claim 7, wherein when the firstsequencing primers are used in step d), said second sequencing primersare not active.
 13. A method of sequencing nucleic acid, comprising: a)providing i) a plurality of different amplicons immobilized on a firstsurface enclosed in a chamber, and ii) sequencing reagents; b)introducing said sequencing reagents into said chamber; and c)performing sequencing by synthesis.
 14. The method of claim 13, whereinsaid surface is a glass surface.
 15. The method of claim 13, whereinsaid glass surface is a glass chip.
 16. The method of claim 13, whereinsaid glass surface is a glass slide.
 17. The method of claim 13, whereinsaid chamber is sealed by an adhesive gasket, and a second surface. 18.The method of claim 17, wherein said adhesive gasket is sandwichedbetween said first and second surfaces.
 19. The method of claim 13,wherein said sequencing reagents comprise polymerase and a plurality ofsequencing primers.
 20. The method of claim 13, wherein said amplicon isimmobilized in one or more channels.
 21. The method of claim 19, whereinsaid channels are created by said first surface and a second surface.22. A method of sequencing nucleic acid, comprising: a) providing i) aplurality of different amplicons immobilized on a plurality of beads,said beads enclosed in a chamber, said chamber sealed by an adhesivegasket sandwiched between a first surface and a second surface and ii)sequencing reagents; b) introducing said sequencing reagents into saidchamber; and c) performing sequencing by synthesis.
 23. The method ofclaim 22, wherein said first surface is a glass slide.
 24. The method ofclaim 22, wherein said second surface is metal.
 25. A device, comprisinga plurality of different nucleic acid amplicons immobilized in regionson a glass surface, wherein the amplicon within each region ishomogeneous.
 26. The device of claim 25, wherein said regions are inchannels.
 27. The device of claim 26, wherein said channels are definedby a gasket sandwiched between said glass surface and a second surface.28. The device of claim 27, wherein said gasket is an adhesive gasket.29. The device of claim 26, wherein said channels are approximately10-50 microns in diameter and are spaced approximately 10-100 micronsapart.
 30. The device of claim 26, wherein said amplicons areimmobilized on beads in said channels.
 31. The device of claim 25,wherein said glass surface is a glass slide.